HIV-1 Genotyping of the Protease-Reverse Transcriptase and Integrase Genes to Detect Mutations That Confer Antiretroviral Resistance

Abstract

Major advances in antiretroviral (ARV) therapy during the last decade have made HIV-1 infections a chronic, manageable disease. In spite of these significant advancements, ARV drug resistance remains a hurdle for HIV-infected patients who are committed to lifelong treatments. Several commercially marketed and/or laboratory-developed tests (LDT) are available to detect resistance-associated mutations (RAMs) in HIV-1, by genotyping. These genotyping tests mainly comprise polymerase chain reaction (PCR)-amplification and population, nucleotide sequencing (Sanger methodology) of a large part of the protease (PR), reverse transcriptase (RT), and integrase (IN) genes. In this chapter, we describe HIV-1 PR, RT, and IN genotyping on clinical samples (plasma), using the LDT methodology performed at Janssen Diagnostics BVBA, Belgium (JDx), where the PR-RT genotyping is used as input, to generate a CE-marked vircoTYPE™ HIV-1 report while the IN genotyping is performed as a research-use-only (RUO) assay. The complete HIV-1 PR gene (297 bp; 99 amino acids) and a large part of the RT gene (the first 1,200 bp; 400 amino acids) are amplified and sequenced as a single 1,497 bp fragment. Genotyping of the IN gene is performed by amplification and sequencing of the RT-IN region (the last 459 bp; 153 amino acids of RT with the complete 867 bp; 289 amino acids of IN). This methodology allows identification of nucleoside/-nucleotide reverse transcriptase, non-nucleoside reverse transcriptase, protease, and integrase inhibitor (NRTI, NtRTI, NNRTI, PI, INI) RAMs in the PR-RT and IN genes, which allows to predict viral response against current ARV regimens.

Current Perspectives on HIV-1 Antiretroviral Drug Resistance

“…For HIV-1 genotyping, there are several commercial or laboratory-developed tests are available to detect the resistance associated mutations. These tests mainly utilize PCR amplification and nucleotide sequencing of viral genes such as reverse transcriptase, protease, and integrase [ 307 ]. The WHO and International AIDS Society-USA Panel recommends antiretroviral drug testing as a routine management of the viral infection in developing countries.…”

Cross-clade simultaneous HIV drug resistance genotyping for reverse transcriptase, protease, and integrase inhibitor mutations by Illumina MiSeq

“…The use of integrase inhibitors as part of initial antiretroviral therapy is expected to increase because of the recent FDA approval of a new once daily integrase inhibitor (elvitegravir) that can be co-formulated with emtricitabine, tenofovir disoproxyl fumarate and cobicistat as a single pill once daily regimen (STRIBILD) [ 9 ]. Current genotyping methods used to detect drug resistance to integrase inhibitors are limited and typically involve a separate assay from that used to identify drug resistance to reverse transcriptase and protease inhibitors [ 10 , 11 ]. However, due to proximity of the integrase protein-coding region to the reverse transcriptase and protease protein-coding region in the viral genome, it is feasible to incorporate drug resistance genotyping of all three gene products together from a single PCR amplicon.…”

Multidrug-resistant tuberculosis (MDR-TB) and multidrug-resistant HIV (MDR-HIV) syndemic: challenges in resource limited setting

“…Drug-resistance testing (DRT) through HIV genotyping detects clinically significant mutations in the HIV genome, and is a useful tool for assessing resistance among the commonly used ART drugs, particularly NRTIs and NNRTIs 28. The downward trend in our patient’s CD4 count and the occurrence of opportunistic infections raised the suspicion of possible resistance.…”